Synthesis, in vitro metabolism, cell transformation, mutagenicity, and DNA adduction of dibenzo[c,mno] chrysene

Article abstract of DOI:10.1021/tx0200111

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants. Due to its structural similarity with the potent carcinogen dibenzo [a,l] pyrene (DB [a,l]P) and because of its environmental presence, dibenzo[c,mno]chrysene (naphtho[1,2-a]pyrene, N[1,2-a]P) is of considerable research interest. We therefore developed an efficient synthesis of N[1,2-a]P, and examined its in vitro metabolism by male Sprague Dawley rat liver S9 fraction. Its mutagenic activity in S. typhimurium TA 100 and its morphological cell transforming ability in mouse embryo fibroblasts were evaluated. On the basis of spectral analyses, the in vitro major metabolites were identified as the fjord region dihydrodiol trans-9,10-dihydroxy-9,10- dihydro-N[1,2-a]P (N[1,2-a]P-9,10-dihydrodiol), the K-region diols N[1,2-a]P-4,5-dihydrodiol and N[1,2-a]P-7,8-dihydrodiol, and also the 1-, 3-, and 10-hydroxy-N[1,2-a]P; the structure of N[1,2-a]P-9,10-dihydrodiol was also confirmed by independent synthesis. In assays with S. typhimurium TA 100, N[1,2-a]P-9,10-dihydrodiol was half as mutagenic as (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-dihydrodiol) at ≥ nmol/plate. N[1,2-a]P-9,- 10-dihydrodiol was much more mutagenic than N[1,2-a]P at all dose levels, suggesting that the N[1,2-a]P-9,10-dihydrodiol is the likely proximate mutagen of N[1,2-a]P. Evaluation of morphological cell transformation in C3H10T1/2C18 mouse embryo fibroblasts revealed that N[1,2-a]P was comparable to B[a]P. We further examined the pattern of in vitro adduct formation between calf thymus DNA and (±)-anti-9,10-dihydroxy-9,10-dihydro-11,12-epoxy- 9,10,11,12-tetrahydro-N[1,2-a]P (N[1,2-a]PDE) and found that dG-adduct formation is 2.9-fold greater than dA-adduct formation. On the basis of our results and those reported in the literature, our working hypothesis is that N[1,2-a]P may be added to the list of potent carcinogens that includes DB [a,l]P. This hypothesis is currently being tested in our laboratory.

Full text of DOI:10.1021/tx0200111

9
64
Chem. Res. Toxicol. 2002, 15, 964-971
Syn th esis, in Vitr o Meta bolism , Cell Tr a n sfor m a tion ,
Mu ta gen icity, a n d DNA Ad d u ction of
Diben zo[c,m n o]ch r ysen e
,
†
†
†
†
Dhimant Desai,* Arun K. Sharma, J yh-Ming Lin, J acek Krzeminski,
‡
†
‡
†
Maria Pimentel, Karam El-Bayoumy, Stephen Nesnow, and Shantu Amin
American Health Foundation, 1 Dana Road, Valhalla, New York 10595, and U. S. Environmental
Protection Agency, ORD, MD-68, Research Triangle Park, North Carolina 27711
Received February 6, 2002
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants. Due to
its structural similarity with the potent carcinogen dibenzo[a,l]pyrene (DB[a,l]P) and because
of its environmental presence, dibenzo[c,mno]chrysene (naphtho[1,2-a]pyrene, N[1,2-a]P) is
of considerable research interest. We therefore developed an efficient synthesis of N[1,2-a]P,
and examined its in vitro metabolism by male Sprague Dawley rat liver S9 fraction. Its
mutagenic activity in S. typhimurium TA 100 and its morphological cell transforming ability
in mouse embryo fibroblasts were evaluated. On the basis of spectral analyses, the in vitro
major metabolites were identified as the fjord region dihydrodiol trans-9,10-dihydroxy-9,10-
dihydro-N[1,2-a]P (N[1,2-a]P-9,10-dihydrodiol), the K-region diols N[1,2-a]P-4,5-dihydrodiol
and N[1,2-a]P-7,8-dihydrodiol, and also the 1-, 3-, and 10-hydroxy-N[1,2-a]P; the structure of
N[1,2-a]P-9,10-dihydrodiol was also confirmed by independent synthesis. In assays with S.
typhimurium TA 100, N[1,2-a]P-9,10-dihydrodiol was half as mutagenic as (()-trans-7,8-
dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-dihydrodiol) at g4 nmol/plate. N[1,2-a]P-9,-
1
0-dihydrodiol was much more mutagenic than N[1,2-a]P at all dose levels, suggesting that
the N[1,2-a]P-9,10-dihydrodiol is the likely proximate mutagen of N[1,2-a]P. Evaluation of
morphological cell transformation in C3H10T1/2C18 mouse embryo fibroblasts revealed that
N[1,2-a]P was comparable to B[a]P. We further examined the pattern of in vitro adduct
formation between calf thymus DNA and (()-anti-9,10-dihydroxy-9,10-dihydro-11,12-epoxy-
9
,10,11,12-tetrahydro-N[1,2-a]P (N[1,2-a]PDE) and found that dG-adduct formation is 2.9-
fold greater than dA-adduct formation. On the basis of our results and those reported in the
literature, our working hypothesis is that N[1,2-a]P may be added to the list of potent
carcinogens that includes DB[a,l]P. This hypothesis is currently being tested in our laboratory.
In tr od u ction
Polycyclic aromatic hydrocarbons (PAHs) are envi-
their environmental ubiquity and structural similarity
with DB[a,l]P, some naphthopyrene (NP) isomers have
been synthesized and tested; while N[2,1-a]P and N[2,3-
a]P were moderately active carcinogens, N[2,3-e]P had
no effect (7-9).
1
ronmental pollutants of widely varying structural com-
position. The genotoxicity of PAHs is strongly linked to
their structural features. Dibenzo[a,l]pyrene (DB[a,l]P),
a hexacyclic PAH with a pyrene moiety and a fjord region,
has attracted much attention because of its unsurpassed
mutagenicity and carcinogenicity (1-5). In both mutage-
nicity and carcinogenicity assays, DB[a,l]P is much more
potent than benzo[a]pyrene (B[a]P) (1-5). It is logical,
therefore, to investigate peri-condensed hexacyclic PAHs
with a fjord region with respect to their genotoxicity.
However, peri-condensed hexacyclic PAHs are a diverse
group of compounds with 15 possible different structures,
Out of 12 possible hexacyclic isomers of pyrene, 10 were
known to be present in environmental samples (9);
however, recent studies have also identified the remain-
ing 2 isomers, N[1,2-a]P and naphtho[1,2-e]pyrene [N[1,2-
e]P in coal tar extract, in air-borne particulate matter,
and in marine sediment (Stephen Wise et al., unpub-
lished results)]. We hypothesized that N[1,2-a]P and
N[1,2-e]P are potent genotoxins because of their shared
structural similarity with DB[a,l]P, namely, a pyrene
moiety as well as a fjord region. To test this hypothesis,
we have synthesized N[1,2-a]P and examined its in vitro
metabolism with S9 fraction of phenobarbital/â-naph-
thoflavone-induced male Sprague Dawley rat liver ho-
mogenate. To gain insight into metabolic pathways of
N[1,2-a]P that may lead to its genotoxicity, we evaluated
the mutagenicity of N[1,2-a]P and of its terminal ring
dihydrodiol toward S. typhimurium TA 100. The cyto-
toxicity and morphological cell transforming activity of
N[1,2-a]P were also compared to B[a]P in C3H10T1/2
mouse embryo fibroblast cells (C3H10T1/2).
1
2 of which have a pyrene moiety (6). On the basis of
*
Correspondence should be addressed to this author. Telephone:
(
914) 789-7298; E-mail: dhimant99@hotmail.com.
†
American Health Foundation.
U. S. Environmental Protection Agency.
Abbreviations: PAHs, polycyclic aromatic hydrocarbons; DB[a,l]P,
‡
1
dibenzo[a,l]pyrene; N[1,2-a]P, dibenzo[c,mno]chrysene (naphtho[1,2-
a]pyrene); NP, naphthopyrene; N[1,2-a]P-9,10-dihydrodiol, (()-trans-
9
,10-dihydroxy-9,10-dihydro-N[1,2-a]P; B[a]P-7,8-dihydrodiol, (()-
trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene; N[1,2-a]PDE, (()-anti-
9
,10-dihydroxy-9,10-dihydro-11,12-epoxy-9,10,11,12-tetrahydro-N[1,2-
a]P; N[1,2-e]P, naphtho[1,2-e]pyrene; B[a]P, benzo[a]pyrene; B[c]PhDE,
benzo[c]phenanthrene diol epoxide; DMBA, 7,12-dimethylbenz[a]an-
thracene.
1
0.1021/tx0200111 CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/20/2002

Metabolic Activation of Dibenzo[c,mno]chrysene
Chem. Res. Toxicol., Vol. 15, No. 7, 2002 965
The formation of PAH-DNA adducts is known to be
influenced by the molecular structure of the PAHs. For
example, B[a]P diol epoxide, a planar molecule, reacts
with calf-thymus DNA in vitro to yield exclusively the
B[a]P-dG adduct (10, 11). However, benzo[c]phenan-
threne diol epoxide (B[c]PhDE), a nonplanar molecule,
reacts with calf-thymus DNA to give a mixture of B[c]-
Ph-dG and B[c]Ph-dA adducts (9). The relative ratios of
dA/dG adducts formed differ depending on the B[c]Ph diol
epoxide isomers used, but the dA/dG ratios are between
sodium, 60 mL) was cooled to -78 °C under N
was added PhLi (1.8 M, 4.0 mL, 7.2 mmol) in cyclohexane/ether
70:30), dropwise, and the mixture was stirred for 30 min. The
2
. To this mixture
(
reaction mixture was then allowed to warm to -30 °C, stirred
for another 30 min, and again cooled to -78 °C. A solution of 2
(0.6 g, 1.9 mmol) in THF (40 mL) was then added dropwise,
and the mixture was allowed to come to room temperature and
left overnight. The reaction was quenched with 1 N HCl; the
mixture was washed several times with water and extracted
with ethyl acetate (3 × 25 mL). The combined organic layers
4
were dried (MgSO ), filtered, and concentrated. The resulting
1
.4 and 2.0 in favor of the B[c]Ph-dA adducts (9). This is
residue was purified on a silica gel column by elution with
hexane/ethyl acetate (98:2) to yield a mixture of cis- and trans-
isomers of olefin 4 (0.52 g, 81%), as a clear viscous oil: H NMR
highly significant because many sterically crowded bay-
region PAH-diol-epoxides that favor the formation of
PAH-dA adducts are generally more potent carcinogens
than their less crowded counterparts. To further verify
this steric effect, we examined the pattern of adduct
formation between calf-thymus DNA and N[1,2-a]PDE
in vitro.
1
δ 3.21 (s, 1.55H, trans-OCH
d, 0.48H, cis-olefin H, J ) 7.2 Hz), 5.43 (d, 0.52H, trans-olefin
H, J ) 12.8 Hz), 5.85 (d, 0.48H, cis-olefin H, J ) 7.2 Hz), 7.00
3 3
), 3.72 (s, 1.45H, cis-OCH ), 4.81
(
(
(
(
d, 0.52H, trans-olefin H, J ) 12.8 Hz,), 7.34-7.55 (m, 3H), 7.61
d, 0.5H, cis/trans, J ) 7.5 Hz), 7.80-7.84 (m, 1H), 7.93-7.95
m, 1H), 7.98-8.05 (m, 2H), 8.13-8.25 (m, 5H), 8.35 (d, 0.5H,
+
cis/trans, J ) 7.8 Hz); MS m/z 334 (M ).
Ma ter ia ls a n d Meth od s
1-[2-(â-Met h oxyet h en yl)-4-m et h oxyp h en yl]p yr en e (5).
Olefin 5 was prepared by following a procedure similar to that
used for compound 4, namely, reacting anhydrous (methoxy-
methyl)triphenylphosphonium chloride (21.4 g, 62.5 mmol) in
Syn th esis. Melting points were recorded on a Fisher-J ohnson
melting point apparatus and are uncorrected. Unless stated
otherwise, proton NMR spectra were recorded in a Bruker AM
2
freshly distilled Et O (400 mL) with PhLi (1.8 M, 26 mL, 46.9
3
60WB instrument using CDCl
3
as solvent. The chemical shifts
mmol) in cyclohexane/ether (70:30), and 3 (4.2 g, 12.5 mmol) in
THF (300 mL). The resulting crude olefin 5 was purified on a
silica gel column. Elution with hexane/ethyl acetate (97:3)
are reported in ppm downfield from TMS. MS analyses were
carried out with a Hewlett-Packard model 5988A instrument.
High-resolution MS analyses were determined on a Finnigan
Mat95 instrument, at the University of Minnesota. Thin-layer
chromatography (TLC) was developed on aluminum-supported,
precoated silica gel plates (EM Industries, Gibbstown, NJ ). All
starting materials were obtained from Aldrich Chemical Co.
yielded a mixture of cis- and trans-isomers of olefin 5 (3.83 g,
1
84%): H NMR δ 3.19 (s, 1.65H, trans-OCH
3
), 3.72 (s, 1.35H,
), 3.95 (s, 1.65H, trans-
), 4.76 (d, 0.45H, cis-olefin H, J ) 7.2 Hz), 5.38 (d, 0.55H,
cis-OCH
3 3
), 3.94 (s, 1.35H, cis-OCH
OCH
3
trans-olefin H, J ) 13.1 Hz), 5.85 (d, 0.45H, cis-olefin H, J )
7.2 Hz), 6.89-6.93 (m, 1H), 6.98 (d, 0.55H, trans-olefin H, J )
12.8 Hz), 7.11 (d, 0.55H, trans-H3′, J ) 2.6 Hz), 7.28-7.32 (m,
1H, H6′), 7.79-7.83 (m, 1H), 7.89 (d, 0.45H, cis-H3′, J ) 2.0
Hz), 7.90-7.93 (m, 1H), 7.96-8.04 (m, 2H), 8.91-8.23 (m, 5H);
(Milwaukee, WI) unless stated otherwise. 1-Pyreneboronic acid
(
1) was prepared in quantitative yield by treating 1-bromo-
pyrene with triisopropylborate in the presence of n-butyllithium
as reported in the literature (12). Benzo[c]chrysene-5-carboxy-
aldehyde (12) was prepared as reported earlier (13).
+
MS m/z 364 (M ).
1
-(2-F or m ylp h en yl)p yr en e (2). A mixture of 1-pyrenebo-
ronic acid (1) (1.08 g, 4.4 mmol), 2-bromobenzaldehyde (0.74 g,
.0 mmol), cesium fluoride (1.34 g, 8.8 mmol), and Pd(PPh
0.18 g, 0.16 mmol) in anhydrous DME (30 mL) was heated
under reflux for 20 h. The mixture was brought to room
temperature, and the reaction was quenched with ice-cold H O.
The aqueous layer was extracted with CH Cl
(3 × 25 mL), and
the combined organic layers were washed with H O, dried
MgSO ), and evaporated to give a crude mixture. This mixture
Na p h th o[1,2-a ]p yr en e (N[1,2-a ]P ) (6). To a solution of
olefin 4 (0.4 g, 1.2 mmol) in anhydrous CH
added CH SO H (5 mL) dropwise under N , and the mixture
was stirred at room temperature for 2 h. It was then poured
into ice-cold H O, and the organic layer was separated, and
washed successively with 10% NaHCO solution and several
times with water. The organic layer was dried (MgSO ) and
2 2
Cl (30 mL) was
4
(
3
)
4
3
3
2
2
2
3
2
2
4
2
evaporated, and the crude residue was purified on a silica gel
column with hexane/ethyl acetate (99:1) to give 6 (0.33 g, 91%),
as a pale yellow crystalline solid; mp 166-167 °C, lit (14, 15)
mp 216-218 °C: 1H NMR δ 7.68-7.81 (m, 2H, H10 and H11),
7.96 (d, 1H, J ) 8.9 Hz), 8.02-8.22 (m, 6H), 8.29 (d, 1H, J )
7.9 Hz), 8.34 (d, 1H, H14, J 13,14 ) 9.2 Hz), 8.57 (s, 1H, H6), 9.27
(d, 1H, H12, J 11,12 ) 8.5 Hz), 9.44 (d, 1H, H13, J 13,14 ) 9.2 Hz);
(
4
was purified by silica gel column chromatography with hexane/
ethyl acetate (99:1) as an eluent to give 2 (0.90 g, 73%) as a
pale yellow crystalline solid, mp 118-119 °C: H NMR δ 7.59
d, 1H, J ) 7.54 Hz), 7.63-7.81 (m, 1H), 7.75-7.80 (m, 2H),
.95 (d, 1H, J ) 7.87 Hz), 8.02-8.07 (m, 2H), 8.15 (s, 2H), 8.18-
.20 (m, 2H), 8.24-8.27 (m, 2H), 9.66 (s, 1H, CHO); MS m/z
06 (M ).
1
(
7
8
3
high-resolution MS m/z calcd for C24H14, 302.1096; found,
302.1093.
+
1
-(2-F or m yl-4-m eth oxyp h en yl)p yr en e (3). In a similar
manner, reacting 1-pyreneboronic acid (1) (3.60 g, 14.6 mmol),
-bromo-5-methoxybenzaldehyde (2.86 g, 13.3 mmol), CsF (4.44
g, 29.3 mmol), and Pd(PPh (0.61 g, 0.53 mmol) in anhydrous
10-Meth oxy-N[1,2-a ]P (7). By following a procedure identi-
cal to that used above, the methoxy derivative 7 was prepared
2
by reacting olefin 5 (3.64 g, 10 mmol) in anhydrous CH
2 2
Cl (50
3
)
4
mL) and CH SO H (25 mL). The crude residue was purified on
3
3
DME (100 mL) gave crude 3 which was purified on a silica gel
a silica gel column with hexane/ethyl acetate (97:3) to give 7
column with hexane/ethyl acetate (98:2) as an eluent to yield
(2.3 g, 77%) as a pale yellow crystalline solid, mp 192-193 °C:
1
4
2
J
.1 g (92%) of aldehyde 3 as a pale yellow crystalline solid, mp
3
H NMR δ 4.05 (s, 3H, OCH ), 7.39 (dd, 1H, H11, J 9,11 ) 2.6
1
01-202 °C: H NMR δ 3.99 (s, 3H, OCH
3
), 7.33 (dd, 1H, H5′,
Hz, J 11,12 ) 9.2 Hz), 7.46 (d, 1H, H9, J 9,11 ) 2.6 Hz), 7.88 (d,
1H, J ) 8.9 Hz), 8.01-8.18 (m, 5H), 8.26 (d, 1H, J ) 7.9 Hz),
8.29 (d, 1H, H14, J 14,13 ) 9.2 Hz), 8.53 (s, 1H, H6), 9.16 (d, 1H,
H12, J 12,11 ) 9.2 Hz), 9.34 (d, 1H, H13, J 13,14 ) 9.2 Hz); high-
5′,3′ ) 2.9 Hz and J 5′,6′ ) 8.2 Hz), 7.50 (d, 1H, H6′, J 6′,5′ ) 8.2
Hz), 7.67 (d, 1H, H3′, J 3′,5′ ) 2.9 Hz), 7.79 (d, 1H, H9, J 9,10 ) 9.2
Hz), 7.93 (d, 1H, H3, J 2,3 ) 7.5 Hz), 8.01-8.06 (m, 2H, H7 and
H10), 8.14 (s, 2H, H4 and H5), 8.19 (d, 1H, H8, J 7,8 ) 7.87),
resolution MS m/z calcd for C25
0-Hyd r oxy-N[1,2-a ]P (8). To a stirred solution of 10-
methoxy-N[1,2-a]P (7) (2.09 g, 6.03 mmol) in CH Cl (180 mL)
at room temperature was added dropwise a solution of BBr (1
M solution in CH Cl , 12.6 mL, 12.6 mmol) under N over 10
min. After continuous stirring at room temperature for 5 h, the
H16O, 332.1199; found, 332.1193.
8
.24 (d, 2H, H2 and H6, J 2,3 ) 7.5 Hz), 9.61 (s, 1H, CHO); high-
resolution MS m/z calcd for C24 , 336.1153; found, 336.1150.
-[2-(â-Meth oxyeth en yl)p h en yl]p yr en e (4). A suspension
of anhydrous (methoxymethyl)triphenylphosphonium chloride
3.3 g, 9.6 mmol) in freshly distilled diethyl ether (dried over
1
16 2
H O
2
2
1
3
2
2
2
(

9
66 Chem. Res. Toxicol., Vol. 15, No. 7, 2002
Desai et al.
reaction was quenched with ice-cold H
washed several times with water, and dried over MgSO
Removal of the solvent gave the crude solid that was purified
by column chromatography with hexane/ethyl acetate (10:1) to
2
O. The organic layer was
triphenylphosphonium chloride (21.4 g, 62.5 mmol), PhLi (1.8
M, 26 mL, 46.9 mmol), and aldehyde 12 (3.8 g, 12.5 mmol). The
crude product 13 was purified on a silica gel column by elution
with hexane/ethyl acetate (97:3) to yield a mixture of the major
4
.
yield 8 (1.36 g, 68%) as a pale yellow crystalline solid, mp 216-
trans-isomer and a minor cis-isomer of olefin 13 (3.43 g, 82%):
1
1
2
2
1
8
18 °C: H NMR δ 5.13 (s, 1H, OH), 7.32 (dd, 1H, H11, J 9,11
)
H NMR δ 3.88 (s, 2.85H, trans-OCH
3
), 3.90 (s, 0.15H, cis-
.9 Hz, J 11,12 ) 9.2 Hz), 7.45 (d, 1H, H9, J 9,10 ) 2.9 Hz), 7.83 (d,
H, J ) 8.9 Hz), 8.02-8.08 (m, 3H), 8.13 (d, 1H, J ) 8.9 Hz),
.18 (d, 1H, J ) 7.5 Hz), 8.27 (d, 1H, J ) 6.9 Hz), 8.30 (d, 1H,
OCH
3
), 6.10 (d, 0.05H, cis-olefin H, J ) 7.2 Hz), 6.50 (d, 0.05H,
cis-olefin H, J ) 7.2 Hz), 6.71 (d, 0.95H, trans-olefin H, J )
12.5 Hz), 7.21 (d, 0.95H, trans-olefin, J ) 12.5 Hz), 7.58-7.69
(m, 4H, H2, H3, H10, and H11), 7.82-7.91 (m, 4H, H6, H7, H8,
and H14), 7.91-8.03 (m, 2H, H1 and H9), 8.90 (d, 1H, H12, J 12,11
H14, J 13,14 ) 9.2 Hz), 8.54 (s, 1H, H6), 9.15 (d, 1H, H12, J 12,11
.2 Hz), 9.33 (d, 1H, H13, J 13,14 ) 9.2 Hz); high-resolution MS
m/z calcd for C24 14O, 318.1045; found, 318.1045.
N[1,2-a ]P -9,10-d ion e (9). To a stirred suspension of the
hydroxy derivative, 8 (1.24 g, 3.9 mmol) in a mixture of CH
Cl /C /THF (60:180:6) (246 mL) and Adogen 464 (3 drops),
was added an aqueous solution of KH PO (0.17 M, 210 mL)
and Fremy’s salt (3.14 g, 11.7 mmol). The reaction mixture was
stirred at room temperature for 3 h and then diluted with CH
Cl
. The organic layer was separated, washed with water (3 ×
0 mL), dried (MgSO ), and filtered. The crude residue obtained
)
9
H
) 7.5 Hz), 8.91 (d, 1H, H13, J 13,14 ) 9.2 Hz), 9.19 (d, 1H, H4,
+
J
3,4 ) 7.9 Hz); MS m/z (relative intensity) 334 (M , 60), 303
+
(M -OCH3, 100), 289 (50).
2
-
2
6
H
6
Na p h th o[1,2-a ]p yr en e (N[1,2-a ]P ) (6). In a nitrogen at-
2
4
mosphere, to a solution of olefin 13 (3.34 g, 10 mmol) in
anhydrous CH Cl (50 mL) was added dropwise CH SO H (25
mL), and the mixture was stirred at room temperature for 3 h.
It was then poured into ice-cold H O, and the organic layer was
separated and washed successively with 10% NaHCO solution
and several times with water. The organic layer was dried
(MgSO ) and evaporated, and the crude residue was purified
2
2
3
3
2
-
2
2
5
4
3
after removal of the solvent was washed several times with a
mixture of hexane/acetone (8:2) to give 9 (0.97 g, 75%) as a dark
4
1
red solid, mp 190-191 °C: H NMR δ 6.51 (d, 1H, H11, J 11,12
)
on a silica gel column with hexane/ethyl acetate (97:3) as an
eluent to give 6 (2.1 g, 69%) as a pale yellow crystalline solid,
mp 166-167 °C.
1
0.5 Hz), 7.95 (d, 1H, J ) 8.9 Hz), 8.00 (d, 1H, J ) 6.9 Hz), 8.06
t, 1H, H2, J ) 7.8 Hz), 8.17 (d, 1H, J ) 6.9 Hz), 8.20 (d, 1H, J
8.5 Hz), 8.28-8.33 (m, 4H), 8.53 (d, 1H, H12, J 11,12 ) 10.5
Hz), 8.56 (d, 1H, H13, J 13,14 ) 8.9 Hz); high-resolution MS m/z
calcd for C24 , 332.0838; found, 332.0837.
()-tr a n s-9,10-Dih yd r oxy-9,10-d ih yd r o-N[1,2-a ]P (10). To
a suspension of dione 9 (0.15 g, 0.45 mmol) in ethanol (300 mL)
was added NaBH (0.51 g, 13.5 mmol) in several portions over
0 min. The mixture was stirred for 48 h at room temperature
(
)
In Vitr o Meta bolism of Na p h th o[1,2-a ]p yr en e w ith
P h en oba r bita l/â-Na p h th ofla von e-In d u ced Ma le Sp r a gu e
Da w ley Ra t S9 Liver Hom ogen a te. N[1,2-a]P (2 mg in 200
µL of DMSO) was incubated at 37 °C for 20 min, in the presence
of cofactors and 8 mL of the 9000g supernatant from livers of
phenobarbital/â-naphthoflavone-induced male Sprague Dawley
rats (16). The mixture was extracted with EtOAc (3 × 30 mL),
12 2
H O
(
4
1
while a stream of oxygen was bubbled through the solution. The
solution was then concentrated under reduced pressure to one-
fourth of its original volume. This concentrate was diluted with
4
dried (MgSO ), filtered, and concentrated at reduced pressure
to give a residue that was dissolved in methanol. The metabo-
lites of N[1,2-a]P were analyzed by HPLC on a 4.6 × 250 mm
(5 µm) Vydac C18 reverse-phase column (Separation Group,
Hesperia, CA) with solvent A (H O) and solvent B (methanol),
2
using a gradient program from A:B (50:50) to A:B (0:100) over
40 min.
H
2
O, and extracted with EtOAc (3 × 30 mL). The organic layer
was dried (Na SO ), filtered, and concentrated, and the resulting
residue was washed with a mixture of hexane/diethyl ether
9:1) to give diol 10. Purification by column chromatography
2
4
(
using methylene chloride with gradually increasing ethyl
acetate gave diol 10 (0.12 g, 79%), mp 225-227 °C: H NMR
Mu t a gen icit y Assa ys. N[1,2-a]P, N[1,2-a]P-9,10-dihydro-
diol, B[a]P, and B[a]P-7,8-dihydrodiol were each dissolved in
DMSO and assayed in S. typhimurium strain TA 100 with 20
min of preincubation according to an established method (17).
In these studies, 50 µL of phenobarbital/â-naphthoflavone-
induced male Sprague Dawley rat liver homogenate S9 mix (75
mg of protein/mL) was used per plate, and sodium azide at a
dose of 5 µg/plate (1008 His-rev/plate) was the positive control.
Values are reported as the mean ( SD (n ) 3). Background
counts for solvent (121 His-rev/plate) have not been subtracted.
1
(
1
(
J
(
1
DMSO-d
1.5 Hz, J OH,9 ) 5.9 Hz), 5.42 (d, 1H, OH, J OH,10 ) 4.9 Hz), 5.77
d, 1H, OH, J OH,9 ) 5.9 Hz), 6.36 (dd, 1H, H11, J 11,12 ) 10.1 Hz,
10,11 ) 1.5 Hz), 7.39 (d, 1H, H12, J 11,12 ) 10.1 Hz), 8.01-8.10
m, 4H), 8.21 (d, 1H, J ) 7.2 Hz), 8.28-8.38 (m, 3H), 8.64 (s,
H, H6), 8.95 (d, 1H, H13, J 13,14 ) 9.19 Hz); high-resolution MS
m/z calcd for C24 , 336.1150; found, 336.1150.
()-a n ti-9,10-Dih yd r oxy-9,10-d ih yd r o-11,12-ep oxy-9,10,-
1,12-tetr a h yd r o-N[1,2-a ]P (11). A solution of dihydrodiol 10
0.10 g, 0.3 mmol) and mCPBA (0.52 g, 3.0 mmol) in freshly
distilled THF (100 mL) was stirred at room temperature under
and monitored by normal-phase HPLC using an analytical
6
) δ 4.59-4.65 (m, 1H, H10), 4.69 (dd, 1H, H9, J 9,10 )
16 2
H O
(
1
(
Mor p h ologica l Cell Tr a n sfor m a tion : Cell Cu ltu r e. Cells
from the mouse embryo fibroblast cell line C3H10T1/2Cl8
(passage 8), derived by Reznikoff et al., were utilized in this
study (18). Cells were incubated at 85% relative humidity in
N
2
Licrosorb Si60 column (5 µm) (E. Merck, Darmstadt, Germany)
with hexane/THF (75:25) in an isocratic program at a flow rate
of 1.9 mL/min. After 7 h of stirring, the mixture was diluted
with 150 mL of diethyl ether, washed with cold 2% NaOH (2 ×
2
an atmosphere containing 5% CO in air at 37 °C. The cultures
were grown in Eagle’s basal medium with Earle’s salts and
L-glutamine, supplemented with 10% heat-inactivated fetal
bovine serum (Grand Island Biological Co., Grand Island, NY).
The cells were checked on a routine basis for mycoplasma
contamination by the Gibco MycoTect assay and were found to
be mycoplasma-free.
1
00 mL) and water (3 × 150 mL), and then dried (K
2 3
CO ),
filtered, and concentrated at room temperature. The concentrate
was washed with a mixture of hexane/diethyl ether (75:25, 50
mL) and dried in vacuo to yield diol epoxide 11 (0.08 g, 76%) as
Mor p h ologica l Cell Tr a n sfor m a tion Assa ys. The mor-
phological cell transformation and cytotoxicity procedures of
Mohapatra et al. (19) for C3H10T1/2 cells were used without
modification. C3H10T1/2 cells were seeded for cytotoxicity
studies (6 dishes per concentration) at 200 cells per 60-mm
plastic Petri dish and for transformation studies (24 dishes per
concentration) at 1000 cells per 60-mm plastic Petri dish in 5
mL of medium. The cells were treated with N[1,2-a]P or B[a]P
dissolved in acetone (25 µL) 24 h after seeding. After a 24-h
exposure, the cells were fed with fresh complete medium
containing 25 µg/mL Garamycin. Seven to ten days after the
treatment, the cytotoxicity dishes were fixed with methanol and
1
a pale, yellow solid, mp 191-193 °C: H NMR (DMSO-d
6
) δ
3
.80 (d, 1H, H11, J 11,12 ) 4.3 Hz), 3.87 (d, 1H, H10, J 9,10 ) 8.3
Hz), 4.76 (d, 1H, H9, J 9,10 ) 8.3 Hz), 4.98 (d, 1H, H12, J 11,12
)
4
1
J
.3 Hz), 8.01-8.14 (m, 4H), 8.23 (d, 1H, J ) 7.2 Hz), 8.36 (d,
H, J ) 8.9 Hz), 8.39 (d, 1H, J ) 8.9 Hz), 8.43 (d, 1H, H14,
14,13 ) 9.2 Hz), 8.68 (s, 1H, H6), 9.06 (d, 1H, H13, J 13,14 ) 9.2
Hz); high-resolution MS m/z calcd for C24
52.1099.
-(â-Meth oxyeth en yl)ben zo[c]ch r ysen e (13). By following
16 3
H O , 352.1097; found
3
5
a procedure similar to that used for compound 4, benzo[c]-
chrysene derivative 13 was prepared by using (methoxymethyl)-

Metabolic Activation of Dibenzo[c,mno]chrysene
Chem. Res. Toxicol., Vol. 15, No. 7, 2002 967
Sch em e 1. Syn th eses of Na p h th o[1,2-a ]p yr en e, tr a n s-Na p h th o[1,2-a ]p yr en e-9,10-d ih yd r o Diol, a n d
a n ti-Na p h th o[1,2-a ]p yr en e-9,10-d ih yd r od iol-11,12-ep oxid e
Sch em e 2. Syn th esis of Na p h th o[1,2-a ]p yr en e
stained with Giemsa. The colonies were then counted. The
medium in the cell transformation dishes was changed weekly.
At confluence, the fetal bovine serum level was reduced to 5%.
At the end of 6 weeks, the dishes were fixed, stained with
Giemsa, and scored for morphological transformation according
to published criteria (20).
P r ep a r a t ion of DNA Ad d u ct s. anti-N[1,2-a]PDE (11) (1
mg) in 1.0 mL of THF was added to a solution of calf thymus
DNA (10 mg) in 10 mL of 10 mM Tris-HCl buffer, pH 7. This
mixture was incubated at 37 °C. The DNA was isolated and
enzymatically hydrolyzed to deoxyribonucleosides as described
in the literature (21). Modified deoxyribonucleosides were
analyzed by HPLC as described below.
P r ep a r a tion of th e Sta n d a r d 2′-d Gu o or 2′-d Ad o Ad -
d u cts fr om Diol Ep oxid e. Approximately 100 mg each of 2′-
dGuo-5′-monophosphate or 2′-dAdo-5′-monophosphate was dis-
solved in 10 mL of 10 mM Tris-HCl buffer (pH 7.0). Then anti-
N[1,2-a]PDE (11) (1 mg) in 1.0 mL of acetone/THF (50:50) was
chloride resulted in the formation of olefin 4 which
cyclized readily in the presence of MeSO H to give N[1,2-
added, and the solution was incubated overnight at 37 °C. The
modified deoxyribonucleotides were separated from unmodified
deoxyribonucleotides on Sep-Pak C18 cartridges. They were then
enzymatically hydrolyzed to the corresponding deoxyribonucleo-
sides (21), and analyzed by HPLC with a Beckman Ultrasphere
ODS 5-µm column (4.6 × 250 mm; reverse phase) using the
3
a]P (6) in 77% yield. Interestingly, the melting point of
N[1,2-a]P thus obtained (166-167 °C) differed from that
reported earlier (216-218 °C) (14,15). This implied that
the product reported earlier (15) was perhaps not N[1,2-
a]P.
following solvent system: 46% MeOH in H
2
O for 10 min,
O over
2
O for 10 min at a flow rate of 1
followed by a linear gradient from 46 to 60% MeOH in H
0 min, then 90% MeOH in H
mL/min.
2
To confirm the structure, we thought it worthwhile to
develop an alternate route for the synthesis of N[1,2-a]P
4
(Scheme 2). Compound 12 on treatment with (meth-
oxymethyl)triphenylphosphonium chloride afforded the
olefin 13 which, on acid-catalyzed cyclization with meth-
anesulfonic acid, gave N[1,2-a]P (6) in quantitative yield.
The N[1,2-a]P thus obtained showed MS, NMR, UV, and
mp identical to those obtained earlier by the Suzuki
coupling reaction and, thus, confirmed the assigned
structure 6. These data were also identical to those
reported recently by S. Kumar (22).
Resu lts a n d Discu ssion
This report describes a new convenient synthesis of
N[1,2-a]P that uses the Suzuki coupling reaction as
shown in Scheme 1. The Suzuki coupling reaction was
recently introduced for the syntheses of PAH ring sys-
tems because it can readily be adapted to a large scale
and gives high yields (22-25). The palladium-catalyzed
reaction of 1-pyreneboronic acid (1) and 2-bromobenzal-
dehyde gave compound 2 in 73% yield. Treatment of
aldehyde 2 with (methoxymethyl)triphenylphosphonium
We also describe here the syntheses of the putative
proximate carcinogenic metabolite N[1,2-a]P-9,10-dihy-
drodiol (10) and the ultimate carcinogenic metabolite

9
68 Chem. Res. Toxicol., Vol. 15, No. 7, 2002
Desai et al.
F igu r e 1. HPLC profile of in vitro metabolites of N[1,2-a]P.
Ta ble 1. Meta bolism of N[1,2-a ]P w ith P h en oba r bita l/
â-Na p h th ofla von e-In d u ced Ma le Sp r a gu e Da w ley Ra t
a
Liver Hom ogen a te
peak
number
metabolite identified
1
2
3
4
5
6
7
4,5-dihydroxy-4,5-dihydro-N[1,2-a]P
7,8-dihydroxy-7,8-dihydro-N[1,2-a]P
9,10-dihydroxy-9,10-dihydro-N[1,2-a]P
1- or 3-hydroxy-N[1,2-a]P
10-hydroxy-N[1,2-a]P
1- or 3-hydroxy-N[1,2-a]P
N[1,2-a]P
a
For experimental conditions, see text.
N[1,2-a]PDE (11). The key intermediate 10-methoxy-
N[1,2-a]P (7) was obtained by applying the Suzuki
coupling reaction as depicted in Scheme 1. Demethylation
of 7 with BBr
on oxidation with Fremy’s salt, gave quinone 9. The
reduction of 9 with NaBH in EtOH furnished N[1,2-a]P-
,10-dihydrodiol (10) in 79% yield. Dihydrodiol 10, on
3
afforded 10-hydroxy-N[1,2-a]P (8) which,
4
9
further oxidation with mCPBA, yielded N[1,2-a]PDE (11).
The structures of dihydrodiol 10 and diol epoxide 11 were
assigned on the basis of their H NMR and mass spectra.
F igu r e 2. Proton NMR spectra of N[1,2-a]P metabolites 1-3.
1
The trans structure was assigned to dihydrodiol 10 on
it was assigned as trans-N[1,2-a]P-9,10-dihydrodiol (10).
In a similar fashion, metabolite 5 was identified as 10-
hydroxy-N[1,2-a]P (8), while metabolites 4 and 6 were
identified as 1-hydroxy- and/or 3-hydroxy-N[1,2-a]P based
the basis of the observed coupling constant of 10.5 Hz
1
between protons H9 and H10 in its H NMR spectrum.
The in vitro metabolism study of N[1,2-a]P was carried
out with male Sprague Dawley rat liver S9 fraction
pretreated with phenobarbital/â-naphthoflavone. The
HPLC profile of metabolites is shown in Figure 1, and
the assignments for the metabolites are listed in Table
1
on our analysis of H NMR and mass spectra.
The bacterial mutagenicity assay with S. typhimurium
TA100 is shown in Figure 3A and 3B. B[a]P and B[a]P-
7,8-dihydrodiol were used as positive controls (26). N[1,2-
a]P-9,10-dihydrodiol appears to be a proximate mutagen
since it was more mutagenic than N[1,2-a]P. B[a]P and
N[1,2-a]P showed similar mutagenic activity up to 4
nmol/plate and above 4 nmol/plate, N[1,2-a]P was more
toxic. The corresponding dihydrodiols, namely, B[a]P-7,8-
dihydrodiol and N[1,2-a]P-9,10-dihydrodiol showed simi-
lar mutagenic activity up to 2 nmol/plate; however, at 4
nmol/plate, N[1,2-a]P-9,10-dihydrodiol was half as mu-
tagenic as B[a]P-7,8-dihydrodiol. Dihydrodiol metabolites
were more mutagenic than their corresponding hydro-
1
. Each metabolite was collected from the HPLC and
1
characterized by UV, MS, H and NMR spectroscopic
methods. Assignments of H NMR spectra of metabolites
1
1
-3 are shown in Figure 2. Metabolites were further
confirmed by HPLC co-injection of the synthetic stan-
dards. Metabolites 1 and 2 were identified as 4,5-
dihydroxy-4,5-dihydro-N[1,2-a]P and 7,8-dihydroxy-7,8-
dihydro-N[1,2-a]P, respectively. The spectral properties
of metabolite 3 were identical to those obtained from the
synthetic trans-N[1,2-a]P-9,10-dihydrodiol (10); therefore,

Metabolic Activation of Dibenzo[c,mno]chrysene
Chem. Res. Toxicol., Vol. 15, No. 7, 2002 969
F igu r e 3. (A) Mutagenicity assay of B[a]P and N[1,2-a]P toward S. typhimurium strain TA 100. (B) Mutagenicity assay with B[a]P-
7
,8-dihydrodiol and N[1,2-a]P-9,10-dihydrodiol toward S. typhimurium strain TA 100.
Ta ble 2. Cytotoxicity a n d Mor p h ologica l Cell Tr a n sfor m a tion of C3H10T1/2 Cells by N[1,2-a ]P a n d B[a ]P
Types II&III
foci/dish,
% dishes with
%
survival,
total no. of
total no. of
Type II or III foci,
a
Type II focib
Type III focib
mean ( SDb
b
compound
concn (µM)
mean ( SD
mean ( SD
acetone
N[1,2-a]P
0.5%
0.03
100 ( 0
2
0
4
0
2
0
6
9
24
19
13
1
16
30
0.03 ( 0.04
0.03 ( 0.04
0.07 ( 0.07
0.14 ( 0.11d
0.35 ( 0.20c
0.83 ( 0.43
0.86 ( 0.22c
2.9 ( 4.1
2.8 ( 3.9
7.2 ( 6.9
12.8 ( 9.2
95.3 ( 7.5
c
0
0
0
0
1
3
.09
.19
.31
.99
.99
.31
84 ( 6.5
c
73.8 ( 6.5
4
c
d
68.5 ( 5.7
16
34
43
37
1
32.1 ( 19.2
c
c
65.1 ( 5.5
54.4 ( 14.5
c
c
59.5 ( 8.4
59.7 ( 10.9
c
c
c
51.7 ( 10.7
95.5 ( 0.8
0.69 ( 0.31
50.0 ( 18.9
0.05 ( 0.05e
4.4 ( 4.4
e
B[a]P
0.39
c
c,e
c,e
1
3
.2
.97
81.3 ( 7.0
74.6 ( 3.3
14
24
0.74 ( 0.07
50.2 ( 4.7
c
0.94 ( 0.28c
c
56.5 ( 14.2
a
The mean plating efficiency in untreated C3H10T1/2 cells was 29.9%. The survival data are based on 3 replicate studies; each study
b
consisted of 6 dishes per group plated and scored. Based on 3 replicate studies; each study consisted of 24 dishes per group plated and
c
scored. Statistically different from the corresponding acetone control group at p < 0.05 by the multiple comparison, Bonferroni t-test.
d
One of the 3 replicate studies was significantly different (p < 0.05) from the acetone control while the remaining 2 showed marginal
e
results. Results from two replicate studies.
carbons in TA 100 system. Above 4 nmol/plate N[1,2-a]P-
,10-dihydrodiol was also more toxic than B[a]P-7,8-
dihydrodiol.
The C3H10T1/2 morphological cell transformation
studies indicated that N[1,2-a]P and B[a]P were ap-
proximately similar in transformation activity. Compari-
son of the activity of N[1,2-a]P with that of other potent
transforming agents in C3H10T1/2 cells including DB-
9
Both N[1,2-a]P and B[a]P were cytotoxic to C3H10T1/2
cells as measured by reductions in plating efficiency
[a,l]P, B[a]P, 7,12-dimethylbenz[a]anthracene (DMBA),
(Table 2). In triplicate studies over a concentration range
dibenz[a,h]anthracene, and 3-methylcholanthrene indi-
cated the order of activity of to be: DMBA > DB[a,l]P >
N[1,2-a]P, B[a]P > dibenz[a,h]anthracene > 3-methyl-
cholanthrene (27). DB[a,l]P and DMBA were able to
transform C3H10T1/2 cells at lower concentrations (27).
These results predict the strong potential for N[1,2-a]P
as a tumorigen in vivo because all of the aforementioned
PAHs have been shown to be mouse skin and mouse lung
tumorigens (28).
of 0-3.31 µM, N[1,2-a]P produced a smooth survival
curve to a maximal effect of 51.7% of control. Statistical
significance (p < 0.05) was achieved at equal to or greater
than 0.09 µM. Over an analogous concentration range,
B[a]P produced significant but slightly less cytotoxicity.
Morphological cell transformation measured as the in-
duction of transformed Type II and Type III foci was
determined over the same concentration ranges as the
cytotoxicity studies. In triplicate studies, statistically
significant increases over control (p < 0.05) in the
incidences of dishes exhibiting a Type II or III trans-
formed focus were observed at concentrations of 1.99 and
Fjord region diol epoxides derived from numerous
PAHs are known to favor reaction with dA over reaction
with dG; therefore, we examined the formation pattern
of in vitro adducts with calf-thymus DNA and N[1,2-a]-
PDE. At first, we prepared nucleoside markers from
N[1,2-a]PDE (11) with either 2′-dG-5′-monophosphate or
2′-dA-5′-monophosphate. The modified deoxyribonucle-
otides were enzymatically hydrolyzed to deoxyribonucleo-
sides, which were analyzed by HPLC (panels A and B,
Figure 4). Then, the N[1,2-a]P diol epoxide (11) was
incubated with calf-thymus DNA, and the modified DNA
was isolated and digested enzymatically to give nucleo-
3
.31 µM N[1,2-a]P. Up to 59.7% of the dishes exhibited
a Type II or III focus. B[a]P also produced statistically
significant numbers of dishes with morphologically al-
tered transformed C3H10T1/2 cells (p < 0.05) at 1.2 and
3
.97 µM, giving a maximal level of 56.5% of the dishes
exhibiting a Type II or III focus. The numbers of Type II
and III transformed foci/dish also were highly significant
compared to control (p < 0.05) at concentrations of 1.99
and 3.31 µM N[1,2-a]P and at 1.2 and 3.97 µM B[a]P.

9
70 Chem. Res. Toxicol., Vol. 15, No. 7, 2002
Desai et al.
that of dA-adducts. This is higher than the dG/dA ratio
for 3,4-dihydro-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phen-
anthrene, which is 0.5-0.7, yet it is lower (9) than the
dG/dA ratio of 20 for 7,8-dihydroxy-9,10-epoxy-7,8,9,10-
tetrahydrobenzo[a]pyrene (10). Our observation is con-
sistent with the trend that fjord region diol epoxides
enhance the levels of dA-adducts. It is also in line with
our observation on the adduct formation of 9,10-di-
hydroxy-11,12-epoxy-9,10,11,12-tetrahydrobenzo[c]chry-
sene which yielded 2.6 times more dG-adducts than dA-
adducts (29). Collectively, these results suggest that the
degree of distortion at the crowded fjord region of benzo-
[c]chrysene is similar to that of N[1,2-a]P. On the basis
of our results and those reported in the literature, our
working hypothesis is that N[1,2-a]P may be added to
the list of potent carcinogens that includes DB[a,l]P; this
hypothesis is currently being tested in our laboratory.
Ack n ow led gm en t. This study was supported by NCI
Contract NCI-CB-77022-75 and Cancer Center Support
Grant CA-17613. We thank Ms. Ilse Hoffmann for editing
the manuscript. The research described in this article has
been reviewed by the National Health and Environmen-
tal Effects Research Laboratory, U.S. Environmental
Protection Agency, and approved for publication. Ap-
proval does not signify that the contents necessarily
reflect the views of the Agency nor does mention of trade
names or commercial products constitute endorsement
or recommendation for use.
Refer en ces
(1) Cavalieri, E. L., Higginbotham, S., RamaKrishna, N. V. S.,
Devanesan, P. D., Todorovic, R., Rogan, E. G., and Salmasi, S.
(
1991) Comparative dose-response tumorigenicity studies of
dibenzo[a,l]pyrene versus 7,12-dimethylbenz[a]anthracene, benzo-
a]pyrene, and two dibenzo[a,l]pyrene dihydrodiols in mouse skin
[
and rat mammary gland. Carcinogenesis 12, 1939-1944.
2) Cavalieri, E. L., Rogan, E. G., Higginbotham, S., Cremonesi, P.,
and Salmasi, S. (1989) Tumor-initiating activity in mouse skin
and carcinogenicity in rat mammary gland of dibenzo[a,l]-
pyrenes: the very potent environmental carcinogen dibenzo[a,l]-
pyrene. J . Cancer Res. Clin. Oncol. 115, 67-72.
(
(
3) Durant, J . L., Busby, W. F., Lafleur, A. L., Penman, B. W., and
Crespi, C. L. (1996) Human cell mutagenicity of oxygenated,
nitrated and unsubstituted polycyclic aromatic hydrocarbons
associated with urban aerosols. Mutat. Res. 371, 123-175.
4) Higginbotham, S., RamaKrishna, N. V. S., J ohansson, S. L.,
Rogan, E. G., and Cavalieri, E. L. (1993) Tumor-initiating activity
and carcinogenicity of dibenzo[a,l]pyrene versus 7,12-dimethyl-
benz[a]anthracene and benzo[a]pyrene at low doses in mouse
skin. Carcinogenesis 14, 875-878.
(
(
5) Busby, W. F., Smith, H., Crespi, C. L., and Penman, B. W. (1995)
Mutagenicity of benzo[a]pyrene and dibenzopyrenes in the Sal-
monella typhimurium TM677 and the MCL-5 human cell forward
mutation assays. Mutat. Res. Gen. Toxicol. 342, 9-16.
6) Harvey, R. G. (1991) Polycyclic aromatic hydrocarbons: chemistry
and carcinogenicity, 192 p, Cambridge University Press, Cam-
bridge, U.K.
(
(
(
7) Lacassagne, A., Buu-Hoi, N. P., and Zajdela, F. (1960) Activite
cancerogene d’hydrocarbures polycycliques derives du naph-
thacene. C. R. Acad. Sci. 250, 3547-3548.
8) Dipple, A., Moschel, R. C., and Bigger, C. A. H. (1984) Polycyclic
Aromatic Hydrocarbons. In Chemical Carcinogens (Searle, C. E.,
Ed.) ACS Monograph 182, American Chemical Society, Washing-
ton, DC.
F igu r e 4. HPLC profile of N[1,2-a]P dA, dG, and calf-thymus
DNA adducts.
(9) Dipple, A., Pigott, M. A., Agarwal, S. K., Yagi, H., Sayer, J . M.,
and J erina, D. M. (1987) Optically active benzo[c]phenanthrene
diol epoxides bind extensively to adenine in DNA. Nature 327,
5
35-536.
side adducts. Their HPLC analysis is shown in panel C
of Figure 4. Peaks 1 and 3 were identified as adducts of
dG and peaks 4 and 5 as adducts of dA by comparison of
their retention times to those of the standard markers.
The total adduct formation for dG is 2.9-fold higher than
(10) Cheng, S. C., Hilton, B. D., Roman, J . M., and Dipple, A. (1989)
DNA adducts from carcinogenic and noncarcinogenic enantiomers
of benzo[a]pyrene dihydrodiol epoxide. Chem. Res. Toxicol. 2,
3
34-340.
(
11) Lin, J .-M., Desai, D., Chung, L., Hecht, S. S., and Amin, S. (1999)
Synthesis of anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-

Metabolic Activation of Dibenzo[c,mno]chrysene
Chem. Res. Toxicol., Vol. 15, No. 7, 2002 971
3
1
1-methylbenzo[a]pyrene and its reaction with DNA. Chem. Res.
treated in culture with 7-methylbenz[a]anthracene- H. Cancer
Toxicol. 12, 341-346.
Res. 33, 2378-2385.
(
12) Rice, J . E., and Cai, Z.-W. (1993) An intramolecular arene triflate
coupling reaction for the regiospecific syntheses of substituted
benzofluoranthene. J . Org. Chem. 58, 1415-1424.
13) Desai, D. H., Krzeminski, J ., Lin, J .-M., and Amin, S. (2000)
Syntheses and identification of 5-methylbenzo[c]chrysene me-
tabolites. Proceedings of the 91st Annual Meeting of the American
Association of Cancer Research, p 572, Abstract 3646, San
Francisco, CA.
14) Harvey, R. G. (1997) Polycyclic Aromatic Hydrocarbons, 232 pp,
Wiley-VCH, New York.
15) Zinke, A., Zimmer, W., Ott, R., and Wiesenberger, E. (1951) On
the construction of benzpyrenes from chrysene. Report II: about
(22) Kumar, S. (2001) A convenient and general synthesis of cata- and
peri-condensed polycyclic aromatic hydrocarbons with a fjord-
region. Synthesis, 841-844.
(
(23) Kumar, S. (1998) A new efficient route of the phenolic derivatives
of chrysene and 5-methylchrysene, precursors to dihydrodiol and
diol epoxide metabolites of chrysene and 5-methylchrysene,
through Suzuki cross-coupling reaction, J . Chem. Soc., Perkin
Trans. 1, 3157-3161.
(
24) Zhang, F. L., Cortez, C., and Harvey, R. G. (2000) New synthetic
approach to polycyclic aromatic hydrocarbons and their carcino-
genic metabolites: Derivatives of benzo[s]picene, benzo[rst]pen-
taphene, and dibenzo[b,def]chrysene. J . Org. Chem. 65, 3952-
(
(
3
960.
1
,2,4,5-dibenzpyrene. Monatsh. Chem. 82, 348-358.
(
25) Sharma, A. K., Kumar, S., and Amin, S. (2002) An abbreviated
synthesis of 7,12-dimethylbenz[a]anthracene and benzo[c]chry-
sene metabolites using the Suzuki reaction. Polycyclic Aromat.
Compd. (in press).
26) Wislocki, P., Wood, A. W., Chang, R. L., Levin, W., Yagi, H.,
Hernandez, O., Dansette, P. M., J erina, D. M., and Conney, A.
H. (1976) Mutagenicity and cytotoxicity of benzo[a]pyrene arene
oxides, phenols, quinones, and dihydrodiols in bacterial and
mammalian cells. Cancer Res. 36, 3350-3357.
27) Nesnow, S., Davis, C., Nelson, G., Ross, J . A., Allison, J ., Adams,
L., and King, L. C. (1997) Comparison of the morphological
transforming activities of dibenzo[a,l]pyrene and benzo[a]pyrene
in C3H10T1/2CL8 cells and characterization of the dibenzo[a,l]-
pyrene-DNA adducts. Carcinogenesis 18, 1973-1978.
28) Ross, J . A., and Nesnow, S. (1999) Polycyclic aromatic hydrocar-
bons: correlations between DNA adducts and ras oncogene
mutations. Mutat. Res. 424, 155-166
(
16) Desai, D., Krzeminski, J ., Lin, J .-M., Chaddha, A., Miyata, N.,
Yagi, H., J erina, D. M., and Amin, S. (1999) Synthesis and
identification of benzo[c]chrysene metabolites. Polycyclic Arom.
Compd. 16, 255-264.
17) Maron, D. M., and Ames, B. N. (1983) Revised methods for the
Salmonella mutagenicity test. Mutat. Res. 113, 173-215.
18) Reznikoff, C. A., Brankow, D. W., and Heidelberger, C. (1973)
Establishment and characterization of a cloned line of C3H mouse
embryo cells sensitive to post confluence inhibition of division.
Cancer Res. 33, 3231-3238.
(
(
(
(
(
(
(
19) Mohapatra, N., MacNair, P., Bryant, B. J ., Ellis, S., Rudo, K.,
Sangaiah, R., Gold, A., and Nesnow, S. (1987) Morphological
transforming activity and metabolism of cyclopenta-fused isomers
of benz[a]anthracene in mammalian cells. Mutat. Res. 188, 323-
(
3
34.
20) Reznikoff, C. A., Bertram, J . S., Brankow, D. W., and Heidel-
berger, C. (1973) Quantitative and qualitative studies of chemical
transformation of cloned C3H mouse embryo cells sensitive to post
confluence inhibition of cell division. Cancer Res. 33, 3239-
(
29) Lin, J .-M., Boyiri, T., Krzeminski, J ., Desai, D., El-Bayoumy, K.,
and Amin, S. (2001) Comparative mammary carcinogenicity in
the rat and in vitro DNA adduct formation of bay-and fjord-region
diol epoxides of benzo[c]chrysene (manuscript in preparation).
3
249.
21) Baird, W. M., and Brookes, P. (1973) Isolation of the hydrocarbon-
deoxyribonucleoside products from the DNA of mouse embryo cells
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